25 research outputs found
In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment
Cross-Section Measurement of Virtual Photoproduction of Iso-Triplet Three-Body Hypernucleus, ⋀nn
Missing-mass spectroscopy with the 3H(e, e′K+) reaction was carried out at Jefferson Lab’s (JLab) Hall A in Oct–Nov, 2018. The differential cross section for the 3H(γ∗, K+)Λnn was deduced at ω = Ee − Ee′ = 2.102 GeV and at the forward K+-scattering angle (0° ≤ θγ∗K ≤ 5°) in the laboratory frame. Given typical predicted energies and decay widths, which are (BΛ, Γ) = (−0.25, 0.8) and (−0.55, 4.7) MeV, the cross sections were found to be 11.2 ± 4.8(stat.)+4.1−2.1(sys.) and 18.1 ± 6.8(stat.)+4.2−2.9(sys.) nb/sr, respectively. The obtained result would impose a constraint for interaction models particularly between Λ and neutron by comparing to theoretical calculations
Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression
The utility of CRISPR-based technologies could be enhanced with the ability to control the spatial and temporal expression of gRNAs. Here the authors design a tRNA scaffold for highly specific gRNA production from a Pol II promoter
In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3′UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment. © 2017 The Author(s)1
Discrete-to-analog signal conversion in human pluripotent stem cells
During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein we report the generation of synthetic gene circuits that can detect a discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond
Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells
During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.ISSN:1744-429
The cross-section measurement for the ³H(e, e′K⁺)nnΛ reaction
電荷をもたない奇妙な原子核の高精度探索 --ラムダ-中性子-中性子の三体系--. 京都大学プレスリリース. 2022-03-08.The small binding energy of the hypertriton leads to predictions of the non-existence of bound hypernuclei for isotriplet three-body systems such as nnΛ. However, invariant mass spectroscopy at GSI has reported events that may be interpreted as the bound nnΛ state. The nnΛ state was sought by missing-mass spectroscopy via the (e, e′K⁺) reaction at Jefferson Lab’s experimental Hall A. The present experiment has higher sensitivity to the nnΛ-state investigation in terms of better precision by a factor of about three. The analysis shown in this article focuses on the derivation of the reaction cross-section for the ³H(γ*, K⁺)X reaction. Events that were detected in an acceptance, where a Monte Carlo simulation could reproduce the data well (), were analyzed to minimize the systematic uncertainty. No significant structures were observed with the acceptance cuts, and the upper limits of the production cross-section of the nnΛ state were obtained to be 21 and at the confidence level when theoretical predictions of (−BΛ, Γ) = (0.25, 0.8) MeV and (0.55, 4.7) MeV, respectively, were assumed. The cross-section result provides valuable information for examining the existence of nnΛ
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Publisher Correction: Semaphorin 3A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8 + T cells
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Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.
Acknowledgements: The authors wish to thank members of the Vincenzo Cerundolo and Tudor A. Fulga laboratories, University of Oxford, for helpful discussions and suggestions. Simon Davis and Oliver Bannard, University of Oxford, for helpful advice and guidance. The staff of the University of Oxford Department of Biomedical Services for animal husbandry. Christoffer Lagerholm and Dominic Waithe of the Wolfson Imaging Centre at University of Oxford for microscopy training and support. Helena Coker and Kseniya Korobchevskaya from the Oxford-ZEISS Centre of Excellence in Biomedical Imaging at Kennedy Institute of Rheumatology which is supported by the Kennedy Trust for Rheumatology Research, IDRM and Carl Zeiss GMBH. The Medical Research Council (MRC) WIMM Flow Cytometry Facility for training and support. The Oxford Centre for Histopathology Research and the Oxford Radcliffe Biobank, which are supported by the NIHR Oxford Biomedical Research Centre. Lisa Browning, Oxford University Hospital, for examination of histology samples. David Pinto, University of Oxford, for code for analysis of CDR3 sequences. This work was supported by the U.K. MRC (MRC Human Immunology Unit), the Oxford Biomedical Research Centre, and Cancer Research UK (CRUK) through CRUK Cancer Centre (C399/A2291 to V.C.; C38302/A17319 to V.K.W.; C375/A17721 to E.Y.J.; 29549 to A.G.; CTRQQR-2021\100002 to J.A.B.); the Wellcome Trust (212343/Z/18/Z to M.F.; 100262Z/12/Z to M.L.D. and S.V.) and Kennedy Trust for Rheumatology Research (to M.F., M.L.D., S.V., A.G., J.M.M.), European Commission (ERC-2014-AdG_670930 to M.L.D. and V.M.), Cancer Research Institute (to V.M.), and EPSRC (EP/S004459/1 to M.F. and H.C.Y). The Wellcome Centre for Human Genetics is supported by Wellcome Trust Centre grant 203141/Z/16/Z. P.S.M. is supported by a Jean Shanks Foundation/Pathological Society of Great Britain & Ireland Clinical Research Training Fellowship. C.K. is supported by a Wellcome Studentship (105401/Z/14/Z). V.J. is supported by an EMBO Long-Term Fellowship (ALTF 1061–2017). J.A.B. is supported by EPSRC/MRC Centre for Doctoral Training in Systems Approaches to Biomedical Science (EP/G037280/1) and the EPSRC Impact Acceleration Account (EP/R511742/1). L.R.O. is supported by the Independent Research Fund Denmark (8048-00078B). V.K.W. is supported by CRUK Oxford Centre Prize DPhil Studentship. M.B.B. is supported by Early-Career Clinician Scientists fellowship from the Lundbeck Foundation (R381-2021-1278). A.V.H. was supported by a Wellcome Trust Clinical Research Fellowship (106287/Z/14/Z) and an A.G. Leventis Foundation Scholarship. A.V.H. is currently supported by a NIHR/University of Cambridge Clinical Lectureship (RC30016) and a Clinical Lecturer Starter Grant from the Academy Of Medical Sciences (G122195; RDAG/600).Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours